Abstract Despite widespread clinical use, knowledge of the mechanisms of general anesthetics is insufficient to explain how they produce amnesia, unconsciousness or immobilization (with increasing doses), the cardinal features of general anesthesia. The long-term goal of this project is to define the synaptic mechanisms that underlie the actions of general anesthetics on the CNS. Anesthetics have potent and specific effects on synaptic transmission, including both presynaptic actions on the release of neurotransmitters and postsynaptic actions on receptors and dendritic spines. The principal objective of this research proposal is to understand synapse- specific presynaptic effects of volatile anesthetics which are poorly understood. Our central hypothesis is that general anesthetics have synapse-specific mechanisms resulting in selective effects on presynaptic ion channels and exocytosis. The rationale underlying this proposal is that understanding presynaptic anesthetic actions relevant to their therapeutic (unconsciousness, amnesia, immobility) and toxic (neurotoxicity, cognitive dysfunction, respiratory and cardiovascular depression) effects of anesthetics is essential for developing new anesthetics with improved side-effect profiles and for optimizing current anesthetic techniques in increasingly high-risk patients. Our proposal that volatile anesthetics have distinct effects on the release of various neurotransmitters due to differential presynaptic expression of anesthetic-sensitive ion channels coupled to transmitter release, in particular voltage-gated sodium and calcium channels, is innovative in approach and employs recently developed techniques in neuroscience and structural biology. The central hypothesis will be tested using an integrative and collaborative multidisciplinary approach by the following three specific aims employing in vivo, cellular and molecular methods: 1) Identify nerve terminal-specific presynaptic mechanisms that influence the sensitivity of synaptic vesicle exocytosis to volatile anesthetics to test the hypothesis that volatile anesthetics differentially inhibit synaptic vesicle exocytosis by nerve terminal-specific mechanisms resulting from heterogeneous presynaptic ion channel expression; 2) Determine the effects of volatile anesthetics on neuronal intracellular Ca2+ regulation and its impact on synaptic vesicle exocytosis to test the hypothesis that volatile anesthetic effects on intracellular Ca2+ dynamics influence synaptic vesicle exocytosis; and 3) Identify volatile anesthetic binding sites on voltage-gated sodium channels using the bacterial homologue NavMs to test the hypothesis that volatile anesthetic inhibition involves direct interactions. The research is significant in applying multidisciplinary and complementary electrophysiological, biophysical, and imaging approaches involving a team of expert collaborators. The expected outcome is a molecular understanding of synaptic anesthetic mechanisms underlying desirable and potentially toxic anesthetic effects on excitatory and inhibitory synaptic transmission. Our results will have positive impact on the rational use and future development of general anesthetics, an increasingly important class of essential medicines.